Selenocysteine (Sec)-containing proteins (selenoproteins) are rare but widely distributed in all domains of life (Hatfield and Gladyshev, 2002), including bacteria (Bock et al., 2006; Stadtman, 2002), archaea (Rother et al., 2001) and eukaryotes (Lescure et al., 1999; Castellano et al., 2001; Kryukov et al., 2003). The human genome possesses 25 genes encoding such proteins (Kryukov et al., 2003). Table 1 lists the known human selenoproteins along with disclosed functions and/or non-limiting uses for certain members.
TABLE 1Human SelenoproteinsHuman selenoproteinsFunctionsGlutathione peroxidase 1In blood cells, marker of Se nutritionGlutathione peroxidase 2Glutathione peroxidase 3Plasma protein, marker of Se status/nutritionGlutathione peroxidase 4Essential for male reproduction (spermmaturation)Glutathione peroxidase 6
TABLE 1Human SelenoproteinsHuman selenoproteinsFunctionsThioredoxin reductase 1Target for cancer therapy. Severalknown classes of anti-cancerdrugs target this proteinThioredoxin reductase 2Thioredoxin reductase 3Deiodinase 1Thyroid hormone metabolismDeiodinase 2Thyroid hormone metabolismDeiodinase 3Thyroid hormone metabolismMethionine-R-sulfoxide reductaseSelenophosphate synthetase 215-SepHas a role in cancer preventionSelenoprotein HSelenoprotein ISelenoprotein KSelenoprotein MSelenoprotein NMutations lead to muscle disordersSelenoprotein OSelenoprotein PMajor plasma selenoprotein,marker of Se statusSelenoprotein SRole in inflammationSelenoprotein TSelenoprotein VSelenoprotein W
The class of selenoproteins is defined by the occurrence of Sec, the 21st amino acid encoded by the UGA codon. Selenoproteins utilize the high reactivity of Sec which is located in catalytic centers and serves redox function analogous to the functions of redox-active Cys residues (Johansson et al., 2005). In addition to the UGA codon, a cis-acting element is present within selenoprotein genes, which is also essential for recognition of UGA as the Sec codon. This element is a stem-loop structure known as the selenocysteine insertion sequence (SECIS) and is located in coding regions of bacterial genes and in the 3′-UTRs of archaeal and eukaryotic selenoprotein genes (Berry et al., 1991; Low and Berry, 1996).
One principal feature of previously disclosed eukaryotic SECIS elements is a segment comprising four non-Watson-Crick base pairs 5′-UGAN . . . NGAN-3′ referred to as a quartet sequence (Berry et al., 1997; Walczak et al., 1996; Korotkov et al., 2002; Walczak et al., 1998). In previously disclosed eukaryotic SECIS elements, the U residue of the quartet sequence is invariant. Nucleotides comprising the 5′-UGAN . . . NGAN-3′ quartet sequence interact with SECIS-binding protein 2 (SBP2) (Copeland et al., 2000; Low et al., 2000) which can form a complex with the Sec-specific elongation factor, known as EFsec, and tRNA[Ser]Sec (Fagegaltier et al., 2000; Tujebajeva et al., 2000). This protein-RNA complex functions by inserting Sec in response to UGA codons in mRNAs containing SECIS elements in the 3′UTR region (Atkins and Gesteland, 2000). Previously disclosed features of SECIS elements include an unpaired residue, usually an A, immediately preceding the 5′-terminus of the aforementioned 5′-UGAN-3′ quartet sequence (5′-AUGAN-3′) and an unpaired AA or CC motif in a region known as the apical loop. While having low sequence conservation, the secondary structure of eukaryotic SECIS elements is conserved and thermodynamically stable (Martin et al., 1996; Martin et al., 1998). Several algorithms have been developed and successfully applied in genomic searches to identify SECIS stem-loop structures and the associated selenoprotein genes in nucleotide sequence databases (Lescure et al., 1999).
Selenoproteins are notoriously difficult targets for recombinant expression. The bacterial Sec insertion system is different from that in eukaryotes in that the bacterial SECIS is present in the coding region downstream of the Sec codon, whereas the eukaryotic SECIS is in the 3′-UTR. Therefore, expression of recombinant proteins in E. coli requires modification of the coding regions of selenoproteins in the vicinity of their active sites. Furthermore, some selenoproteins can only be expressed in eukaryotes due to unique posttranslational modification requirements of those proteins. In both bacterial and eukaryotic systems, efficiency of Sec insertion into recombinant proteins is typically low as the major products are often the truncated forms of selenoproteins. To overcome this problem, several methods for production of recombinant selenoproteins have been proposed (Eckenroth et al., 2006; Su et al., 2005; Arner et al., 1999; Rengby and Arner, 2007). However, there is still a need for compositions and methods that provide for cost-effective, high yield production of recombinant selenoproteins.